1. Field of the Invention
The invention relates to an improved process for enzymically galactosylating monosaccharides and oligosaccharides, with in-situ regeneration of the nucleotide sugar (or of the nucleoside diphosphate sugar), in the presence of sucrose synthase, .beta.-1-4-galactosyl transferase and uridine diphosphate-glucose 4'-epimerase (UDP-glucose 4'-epimerase).
2. Description of the Related Art
Enzymic syntheses of N-acetyllactosamine (LacNAc) and its derivatives using .beta.-1-4-galactosyl transferase (GalT) EC 2.4.1.38! have been known for a long time (C. H. Wong et al.: J. Am. Chem. Soc. 118, 8137 (1991), J. Org. Chem. 57, 4343 (1992)). Wong et al. (FIGS. 1 and 2) developed LacNAc syntheses which involved in-situ regeneration of UDP-glucose, which made it unnecessary to use stoichiometric quantities of the expensive nucleotide sugars, but which nevertheless required 6 different enzymes.
As compared with the previously known cycles, the LacNAc cycle (FIG. 3) proposed by Elling et al. (Glycobiology 3, 349 (1993), DE 42 21 595 C1)), represents an improvement in so far as only three enzymes, i.e. rice sucrose synthase, .beta.-1-4-galactosyl transferase and UDP-glucose 4'-epimerase, instead of six still have to be used for synthesis. The disaccharides which are synthesized are the starting compounds for further reactions with different transferases, e.g. sialyl transferases and fucosyl transferases. The target products for these enzyme syntheses are sialyl Lewis X and its derivatives (Ichikawa et al. J. Am. Chem. Soc. 114, 9283 (1992)), whose importance in cell/cell recognition is the subject of intensive research (DeFrees et al. J. Am. Chem. Soc. 117, 66 (1995)).
Sucrose synthase EC 2.4.1.13! (S. Syn.), which is a glycosyl transferase which is widely distributed in plants, in particular, and whose function as a catalyst for forming nucleotide sugars in plant metabolism has been summarized by Avigad (in Loewus et al. (Eds) Encyclopedia of Plant Physiology New Series Vol. 13A, Carbohydrates I, Intracellular Carbohydrates, Springer Verlag, Berlin, 217-347, 1982) is suitable for synthesizing nucleotide sugars such as UDP-Glc, dTDP-Glc, ADP-Glc, CDP-Glc and GDP-Glc (Elling et al. Glycobiology 3, 349 (1993)). The purification of rice sucrose synthase, and its use for the in-situ regeneration of UDP-glucose, have been described by Elling at al. (DE 42 21 595 C1, Biotechnol. Appl. Biochem. 21, 29 (1994)). The rice enzyme is a homotetrameric protein having a molecular weight of 362 kDa. The enzyme has already been used by Zervosen et al. (Angew. Chem. 106, 592 (1994)) for the preparative synthesis of dTDP-Glc in an enzyme membrane reactor (EMR) and employing dTDP as the starting compound.
The central enzyme for LacNAc synthesis is .beta.-1-4-galactosyl transferase, which transfers UDP-galactose to N-acetylglucosamine. This results in N-acetyllactosamine. A number of other monosaccharides and oligosaccharides can be used as acceptors.
The third enzyme in the Elling at al. (DE 42 21 595 C1) LacNAc cycle is UDP-glucose 4'-epimerase E.C. 5.1.3.2!. The Saccharomyces cerevisiae enzyme, which can be purchased from Sigma, is composed of two subunits to which a molecule of NAD is firmly, but not covalently, bound (Fucusawa et al. J. Biol. Chem. 255, 2705 (1980)). This enzyme does not therefore require any externally added cofactor. The properties of the E. coli and yeast epimerases have been described by Frey et al. (in D. Dolphin et al (Eds.) Pyridine Nucleotide Coenzymes: Chemical, Biochemical and Medicinal Aspects, Vol. 2B, Wiley, N.Y. 462-511).
The epimerization is effected by the UDP-glucose being oxidized to the UDP-4'-ketopyranose and the latter subsequently being reduced to the C4' epimer of the starting compound (FIG. 4).
The epimerase is reductively inactivated in the presence of specific sugars, in the presence of UMP or UDP and by the substrates UDP-glucose and UDP-galactose (Carmenes et al. Yeast 2, 101 (1986) Nelestuen et al., J. Biol. Chem. 4, 7533 (1971)). By binding to the enzyme, uridine nucleotides, such as UMP, induce a conformational change which increases the reactivity of bound NAD to reducing substances. The epimerase-NADH which is subsequently formed exhibits only 10-15% of the activity of the native enzyme (Kalckar et al., Proc. Nat. Ac. Sci. 65, 1113 (1970)).
Owing to the epimerase being inactivated, the previously described enzymic syntheses of LacNAc, involving in-situ regeneration of the nucleotide sugars, only achieve low cycle numbers and, particularly in the case of unnatural substrates, only unsatisfactory yields. In order to increase the yields of disaccharide or oligosaccharide it is essential to meter in further epimerase repeatedly, thereby making the synthesis uneconomical.